The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants.
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Where expression in specific tissues or organs is desired, tissue-preferred promoters may be used. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. In contrast, where gene expression in response to internal and external stimulus is desired, an inducible promoter is the regulatory element of choice. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
Frequently it is desirable to express a DNA sequence in response to particular environmental stimuli such as wounding resulting from insect herbivory, and in only those crop locations or plants where the expression product is required. For example, increased resistance of a plant to infestation by insect pathogens might be accomplished by genetic manipulation of the plant""s genome to comprise an inducible promoter operably linked to a heterologous insect-resistance gene such that insect-resistance proteins are produced only in plants being attacked by the insects and only in response to wounding due to insect herbivory.
Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant""s tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise an inducible promoter operably linked to an antisense nucleotide sequence, such that expression of the antisense sequence produces an RNA transcript that interferes with translation of the MRNA of the native DNA sequence.
Disease in plants is caused by biotic and abiotic causes. Biotic causes include fungi, viruses, bacteria, insects and nematodes. An example of the importance of plant disease is illustrated by phytopathogenic fungi, which cause significant annual crop yield losses as well as devastating epidemics. Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. All of the approximately 300,000 species of flowering plants are attacked by pathogenic fungi; however, a single plant species can be host to only a few fungal species, and similarly, most fungi usually have a limited host range. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Molecular methods of crop protection have the potential to implement novel mechanisms for disease resistance and can also be implemented more quickly than traditional breeding methods. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
One solution to this problem is to use conventional plant breeding practices to produce crops that are resistant to an economically damaging pathogens. However, if there is no known pathogen resistance within the species, a transgenic approach must be taken. With this approach, resistance to pathogens is gained through expression of an anti-pathogenic nucleotide sequence in the plant that proves lethal or undesirable to the invading pathogen.
While pathogen-resistant transgenic crops are now becoming common, most of these products are based on gene expression systems wherein the associated anti-pathogenic sequence is constitutively expressed throughout the plant or continuously expressed in specific tissues. Constitutive expression systems for pathogen resistance present several problems including, but not limited to, yield drag potential, environmental concerns, and health or consumer issues. Yield drag becomes a problem when the plant wastes energy and resources making an anti-pathogenic protein that is not needed when the pathogen attack is mild or non-existent. This is often the case when constitutive promoters are used. Furthermore, there are mounting concerns about the massive release of anti-pathogenic proteins, such as Bacillus thuringiensis (Bt) endotoxins into the environment and the possibility that such a situation would increase the potential of pathogens attaining resistance to the protein due to its prolonged presence at high concentrations. Tissue-preferred expression would provide pathogen refugia and reduce the chance of selection for resistance. In addition, there is also a concern among consumers that plant anti-pathogenic proteins would enter the food chain through constitutive expression of the protein in the agronomically important tissues of crops (e.g., the kernels of corn).
Thus, isolation and characterization of inducible promoters that can serve as regulatory regions for expression of heterologous nucleotide sequences of interest in an inducible manner are needed for genetic manipulation of plants.
Compositions and methods for regulating expression of heterologous nucleotide sequences in a plant are provided. Compositions comprise novel nucleotide sequences for promoters that initiate transcription in an inducible manner, in particular in a wound-inducible, chemical-inducible or pathogen-inducible manner. In one embodiment, a transcriptional initiation region isolated from the plant gene 5C9, a patatin-like gene, is provided. A method for expressing a nucleotide sequence of interest in a plant using the transcriptional initiation sequences disclosed herein is provided. The methods comprise transforming a plant cell with a DNA construct that comprises a nucleotide sequence of interest operably linked to the promoter sequence of the present invention and regenerating a stably transformed plant from the transformed plant cell. In this manner, the promoter sequences are useful for regulating the expression of operably linked nucleotide sequences in an inducible manner.
Downstream from and under the transcriptional initiation regulation of the promoter will be a nucleotide sequence of interest that will provide for modification of the phenotype of the plant. Such modification includes modulating the production of an endogenous product, as to amount, relative distribution, or the like. Other modifications include the production of an exogenous expression product to provide for a novel function or product in the plant.